Stable poliomyelitis vaccines

ABSTRACT

ORALLY ADMINISTRABLE POLIOMYELITIS VACCINE CONTAINING ATTENUATED POLIOMYELITIS VIRUS IS STABILIZED BY TH ADDITION OF A PHOSPHATE BUFFER HAVING A PH FROM 4.5 TO 7.0 AT A CONCENTRATION FROM 0.001 M TO 3.0 M.

United States Patent 3,629,399 STABLE POLIOMYELITIS VACCINES Rudolf Mauler, Cappel, near Marhurg an der Lahn, and Horst Gruschkau, Marbach, near Marburg an der Lahn, Germany, assignors to Behringwerke Aktiengesellschaft, Marburg an der Lahn, Germany No Drawing. Filed Aug. 30, 1968, Ser. No. 756,387 Claims priority, application Germany, Sept. 5, 1967, I 16 17 356.1 Int. Cl. A611 27/00 US. Cl. 424-89 3 Claims ABSTRACT OF THE DISCLOSURE Orally administrable poliomyelitis vaccine containing attenuated poliomyelitis virus is stabilized by the addition of a phosphate buffer having a pH from 4.5 to 7.0 at a concentration from 0.001 M to 3.0 M.

The present invention relates to an improved stable orally administrable poliomyelitis vaccine.

Attenuated poliomyelitis viruses are very unstable and can be stored only in the frozen state. When stored at the usual storage temperature of from 4 to 6 C., the oral poliomyelitis vaccines are, however, rapidly inactivated. Many attempts have, therefore, been made to prepare stable oral poliomyelitis vaccines that are stable at temperatures above 0" C. Thus, the attenuated poliomyelitis viruses have been mixed with ground non-hydrated gelatin and filled into capsules, or applied to sugar cores and dried, or mixed with peptones, or salts of bivalent cations or aluminum salts have been added thereto. Those methods, however, have not been successful in preventing the gradual decrease in activity of oral poliomyelitis vaccines at temperatures of from 4 to 6 C.

Another reason for the decrease in activity is that the attenuated poliomyelitis viruses are adsorbed from the solution onto the walls of glass containers. This adsorp tion is increased by shaking, for example during transport, and may inactivate the vaccine to a large extent within a very short time.

We have now found that, the manufacture of an orally administrable poliomyelitis vaccine can be improved and the above-described disadvantages can be avoided, by adding to attenuated poliomyelitis viruses obtained in known manner.

(a) phosphate buffer having a pH-value of from 4.5 to 7.0, preferably 6.7, in a molarity of from 0.001 to 3.0,

Preferred phosphate buffers are McIlvaine-buifer (disodium hydrogenphosphate/citric acid) or Stirensen-buffer (potassium dihydrogenphosphate/disodium hydrogenphosphate). Other phosphate-containing buffer systems, such as potassium dihydrogenphosphate/sodium hydroxide; disodium hydrogenphosphate/sodium dihydrogenphosphate as well as the buffers of Britton and Robinson, Ff Davies and of Teorell and Stenhagen are likewise use- The use of phosphate buffers or alkali metal ions increases the stability of the poliomyelitis vaccine at temperatures above 0 C. Moreover, the addition of casein, casein hydrolysate or lactalbumin hydrolysate prevents adsorption of the poliomyelitis viruses onto the walls of the vessel or mayeven reverse adsorption that has already taken place.

The following examples serve to illustrate the invention,

but they are not intended to limit it thereto.

The experiments described in the examples were carried out using aqueous suspensions of live attenuated poliomyelitis viruses strain Sabin type I and mixtures of types I, II and III (trivalent vaccine). The experiments were evaluated by titration of the virus content in tissue culture tubes and indicated in TCID (tissue culture infectivity dose 50% For the evaluation dilution series having the factor 10 were prepared from the samples. As dilution medium TCM 199 (tissue culture medium 199) was used (Morgan, J. F., Morton, H. 1., and Parker, R. C., 1950, nutrition of animal cells in tissue culture. I. Initial Studies on a Synthetic Medium. Proc. Soc. Exper. Biol. & Med. 73, 18.). Ten tubes were charged each with 1 milliliter of each dilution step, incubated at 35 C. and the results were evaluated on the seventh day. The virus titers were calculated according to Reed and Muench, Am. J. Hyg. 27, 493 (1938). In the shaking tests, the samples were shaken for 12 to 14 hours about times per minute at an ambient temperature of from +4 to +6 C.

EXAMPLE 1 Trivalent oral poliomyelitis vaccines (types I, II and III) were combined with a phosphate buffer according to S6rensen (pH 6.7) up to a final concentration of 0.5 mol/liter; 1% of lactalbumin hydrolysate, 1% of casein hydrolysate and 5% of casein were added to each sample of this solution. All the samples were shaken together with a control sample for 12 hours at 4 to 6 C. by a mechanical device. Another control sample was left unshaken at the same temperature. After 12 hours, the following results were obtained:

Virus titer (T CIDso) Type I Type II Type III 25x10- s.o 10 x10 2333i: l?;k 2I?1 0.2mm adsorption 92%. 4.0x10 adsorption 50%. 0.4 10 adsorption 75%.

Phosphate butler added, pH 7; 0.5 mol Phosphate buffer (pH 6.7; 0.5 mol) and 1% ollaetalbumm hydrol- 2.7X10 adsorption 0%- 8.2)(10 adsorption 0%. 1.7)(10 adsorption 0%.

ysate added 2.6)(10 adsorption 8.1)(10 adsorption 0%- 1.6 10 adsorption 0%.

""""" i h drol sate Phosphate bufier (pTI 6.7; 0.5 mol) and 5% of casein added 2.4 10 adsorption 4%. 7.8)(10 adsorption 2.5%." 1.5)(10 adsorption 6%.

preferably of 0.3 M, to which casein in a concentration 65 of from 0.01 to 10.0, preferably 5.0%, or lactalbumin hydrolysate or casein hydrolysate in a concentration of from 0.6 to 10.0%, preferably 1.0%, relative to the total amount, is advantageously added, or

(b) alkali metal ions, for example sodium or potassium 7 ions, in a molarity between 0.2 M and the saturation concentration, preferably 2.0 M.

EXAMPLE 2 A trivalent oral poliomyelitis vaccine was prepared containing 1.0% of lactalbumin hydrolysate and 0.3 molar phosphate buffer having a pH of 6.7. Besides, an analogous trivalent oral poliomyelitis vaccine was prepared without using lactalbumin hydrolysate nor phosphate buflier. Both vaccines were stored for 6 months at 4 C. The virus content of both vaccines was determined at the beginning, after 3 months and after 6 months of storage.

VACCINES CONTAINING LACTALBUMIN HYDROLY- SATE AND PHOSPHATE BUFFER Virus titer (TCID Type I Type II Type III Initial value at the beginning of cxpcriment 2.5 10 8.2)(10 1.5)( After 3 months 2. 6X10 8. 1X10 1. 5X10 After 6 months 2. 10 8. 25 1o 1 5x10 Loss after 6 month VACCINES WITHOUT ADDI'IIVES Virus titer (TCIDsO) Type II Type III Type I Initial value at the beginning of expcrinient 2. 4X10 8. 2X10 1. 5X10 After 3montl1s 1.1)(10 2.5)(10 4.0 10 After 6 months 1. 2X 10 8. 0X10 8. 0x10 Loss after 6 months, percent 89. 8 81. 2

This experiment demonstrated that a vaccine containing lactalbumin hydrolysate and phosphate buffer remained stable over the whole observation period of 6 months, whereas a vaccine without these additives had lost almost its whole activity.

EXAMPLE 3 Virus content in TCID (type I) Prior to After Additives shaking shaking Losses No additive (control) fi.3 10 9.0)(10 About 84%. 2 mols of NaCl 6.4)(10 6.0X10 About 6%. 1.5 mols of K01 6.4 10 3.9)(10 About 39%.

This experiment demonstrated that an addition of salts of monovalent cations also considerably reduced the adsorption of the oral poliomyelitis virus to glass.

We claim:

1. An orally administrable poliomyelitis vaccine comprising an immunologically effective attenuated poliomyelitis virus stabilized by the presence of a therapeutically acceptable phosphate bufifer having a pH from 4.5 to 7.0 added at a concentration from 0.001 M to 3.0 M.

2. A vaccine as in claim 1 additionally stabilized by the presence of a member selected from the group consisting of casein added at a concentration of 0.01 to 10.0 percent and a hydrolysate of casein or lactalbumin added at a concentration of 0.6 to 10.0 percent.

3. A vaccine as in claim 1 wherein said buffer is selected from the group consisting of KH PO /NaOH, Na HPO /NaH PO and the buffers of Mcllvaine, Stirensen, Britton-Robinson, Davies, and Teorell-Stenhagen.

References Cited UNITED STATES PATENTS 4/1946 Masucci 42489 8/1964 Hennessen et al. 424-89 OTHER REFERENCES RICHARD L. HUFF, Primary Examiner 

